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. 2021 Dec 6;50(1):411–429. doi: 10.1093/nar/gkab1188

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© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 6. — Staufen1 and Staufen1-dsRBD3 rescues HIV-1 IRES activity in Staufen1 knockout HCT116 and HeLa cells. HCT116 or Staufen1 knockout HCT116 (SKO) cells were transfected, or not, with the Stau1⁵⁵-HA₃ expression plasmid (200 or 650 ng), and cell viability (A) and cell proliferation (B) were analyzed by flow cytometry as indicated in Material and Methods. (C, D) The dl HIV-1 IRES plasmid (300 ng) was cotransfected, or not, with the Stau1⁵⁵-YFP expression construct was transfected into HCT116 or SKO cells. (C) The overexpression of the Stau1⁵⁵-YFP and Stau1⁵⁵-F135A-YFP in transfected HCT116 and SKO cells was confirmed by western blot using an anti-GFP antibody and β-actin as a loading control. (D) RLuc and FLuc activities were measured 24 h post-transfection, and results are presented as RTA. The RTA obtained for HCT116 cells transfected with dl HIV-1 IRES in the absence of Stau1⁵⁵-HA₃, or the mutant protein was set to 100%. Statistical analysis was performed using an unpaired two-tailed t-test (ns, non-significant; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). (E, F) The dl HIV-1 IRES plasmid was cotransfected, or not, with the Stau1⁵⁵-GFP-topaz, mutant Stau1⁵⁵-4K-GFP-topaz or Stau1⁵⁵-dsRBD3-GFP-topaz expression construct into HCT116 or SKO cells. (E) The expression of Stau1⁵⁵-GFP-topaz, Stau1⁵⁵-4K-GFP-topaz, and Stau1⁵⁵-dsRBD3-GFP-topaz was confirmed by western blot using an anti-GFP antibody and β-actin as a loading control. (F) RLuc and FLuc activities were measured 24 h post-transfection, and results are presented as RTA. The RTA obtained for the HCT116 cells that did not overexpress Stau1⁵⁵-recombinant proteins or any of its domains was set to 100%. Statistical analysis was performed using an unpaired two-tailed t-test (ns, non-significant; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). (G, H) HeLa cells were cotransfected with the dl HIV-1 IRES plasmid together with the pe-GFP control plasmid, Stau1⁵⁵-4K-GFP-topaz, Stau1⁵⁵-GFP-topaz, or Stau1⁵⁵-dsRBD3-GFP-topaz expression constructs. (G) Immunofluorescence imaging demonstrating expression and localization of peGFP, Stau1⁵⁵-4K-GFP-topaz, Stau1⁵⁵-GFP-topaz, and Stau1⁵⁵-dsRBD3-GFP-topaz (green) in addition to that of RLuc (red) and Fluc (cyan) in HeLa cells cotransfected with the dl HIV IRES plasmid. (H) Graphical representation of results shown in panel (G), calculating [RTA/GFP] from imaging data using the mean fluorescence intensity (MFI) values obtained for FLuc, RLuc, and GFP. Statistical analysis was performed using a one-way ANOVA with Tukey post-test for multiple comparisons. ** P ≤ 0.01; **** P ≤ 0.0001.